We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent ...bioconjugation. An azide‐modified building block reacts first with an ethynylphosphonite through a Staudinger‐phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine‐selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.
Staudinger—Ready—Go! Ethynylphosphonamidates can be chemoselectively incorporated into a given molecule through a Staudinger‐phosphonite reaction, and they react specifically with cysteine residues on proteins to give thiol adducts that are stable under physiological conditions. This enables the facile fusion of complex molecules to proteins.
The membrane and actin cortex of a motile cell can autonomously differentiate into two states, one typical of the front, the other of the tail. On the substrate-attached surface of Dictyostelium ...discoideum cells, dynamic patterns of front-like and tail-like states are generated that are well suited to monitor transitions between these states. To image large-scale pattern dynamics independently of boundary effects, we produced giant cells by electric-pulse-induced cell fusion. In these cells, actin waves are coupled to the front and back of phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-rich bands that have a finite width. These composite waves propagate across the plasma membrane of the giant cells with undiminished velocity. After any disturbance, the bands of PIP3 return to their intrinsic width. Upon collision, the waves locally annihilate each other and change direction; at the cell border they are either extinguished or reflected. Accordingly, expanding areas of progressing PIP3 synthesis become unstable beyond a critical radius, their center switching from a front-like to a tail-like state. Our data suggest that PIP3 patterns in normal-sized cells are segments of the self-organizing patterns that evolve in giant cells.
Requirements for novel bioconjugation reactions for the synthesis of antibody–drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity ...of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys‐selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non‐engineered monoclonal antibodies through a simple one‐pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate‐linked ADCs have excellent properties for next‐generation antibody therapeutics in terms of serum stability and in vivo antitumor activity.
ADphosphonamidateC: Ethynylphosphonamidate conjugation enables straightforward synthesis of ADCs with excellent linkage stability in serum and promising antitumor activity in vivo. The phosphorous core structure of the labeling reagent enables the attachment of an ethylene glycol motif to increase linkage hydrophilicity.
Antibody drug conjugates (ADCs) are rapidly becoming a cornerstone in targeted therapies, especially for the treatment of cancer. Currently, there are 12 FDA-approved ADCs, eight of which have been ...approved within the last five years, with numerous candidates in clinical trials. The promising clinical perspective of ADCs has led to the development of not only novel conjugation techniques, but also antibody formats, linkers, and payloads. While the majority of currently approved ADCs relies on cytotoxic small molecule warheads, alternative modes of action imparted by novel payloads and non-classical antibody formats are gaining attention. In this review, we summarize the current state of the art of ADC technologies, as well as comprehensively examine alternative payloads, such as toxic proteins, cytokines, PROTACs and oligonucleotides, and highlight the potential of multi-specific antibody formats for the next generation of therapeutic antibody conjugates.
The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to ...hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation.
Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or ...dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.
Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site‐specific ...modification of proteins. Antibody‐oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence‐encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub‐tag® technology in combination with Cu‐catalyzed azide‐alkyne cycloaddition for the site‐specific conjugation of single DNA and PNA strands to an eGFP‐binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence‐specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP‐tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody‐oligonucleotides for reversible immunofluorescence imaging.
Protein‐oligonucleotide conjugates. We present a novel strategy for the efficient and site‐specific generation of nanobody‐oligonucleotide conjugates in a 1 : 1 stoichiometry by Tub‐tag‐mediated conjugation. We show a proof‐of‐concept that these conjugates can readily be used for reversible staining in confocal fluorescence microscopy.
Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of ...phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1β. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1β which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1β mutants defective in H3K9me3 binding were less efficient in phase separationin vitro and failed to accumulate at heterochromatin in vivo. We propose that multivalent interactions of HP1β with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments in vivo.
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